Abstract

There are more than 14,000 new cases of malignant high grade glioma diagnosed each year in the US, and although surgical resection, radiation, and cytotoxic therapies are available, these current treatment methods are not curative. Inhibitor of DNA Binding 2 (Id2), a helix-loop-helix (HLH) protein, functions as a dominant negative inhibitor of basic helix-loop-helix (bHLH) transcription factors and is highly expressed in glioma. Recent data from our laboratory defines a critical role for Id2 in the pathogenesis of a subset of glioma known as proneural glioma. Id2 protein function has been characterized in neural progenitor cells (NPCs) where it acts to suppress differentiation and enhance cell cycle progression. These are two key characteristics that are shared with glioma-derived stem cells. We have identified three phosphorylation sites on Id2 in proliferating NPCs using mass spectrometry, and prepared altered Id2 molecules that cannot be phosphorylated at each of these sites. Id2-/- NPCs expressing WT and phosphorylation-site-ablated Id2 have been prepared to examine the role of Id2 phosphorylation in mediating its interactions with binding partners and in modulating the rate of proteasome-dependent degradation. We found an increased level of Id2 in NPCs expressing a mutant form of Id2 that cannot by phosphorylated when compared with NPCs expressing WT Id2. Moreover, the level of WT Id2 protein decreases when NPCs exit the cell cycle; however, unphosphorylated Id2 resists this degradation and exhibits a longer half-life. Stable isotope labeling by amino acids in cell culture (SILAC) experiments revealed that unphosphorylated Id2 molecules bind significantly less than WT Id2 to several anaphase promoting complex (APC) subunits. These findings suggested a possible mechanism for phosphorylation-dependent proteasome-mediated degradation of Id2 in NPCs. Understanding the effect of Id2 phosphorylation in modulating steady state levels of this important protein will inform future studies designed to identify pathways of interest in the transformation of NPCs and the malignant progression of NPC-initiated glioma. This work was supported in part by the Theodora B. Betz Foundation.